DESCRIPTION: The long-term goal of the proposed research is to understand the molecular mechanisms by which integrins transduce signals across the membrane to regulate cellular functions. The specific focus is on focal adhesion kinase (FAK), a 125 KD tyrosine kinase that was originally identified as an integrin-regulated phosphoprotein in fibroblasts. Although FAK can interact with a variety of intracellular signaling molecules, the relevance of these interactions with regard to FAK s role in intracellular signal transduction by integrin remains poorly defined. This competing renewal application proposes to dissect the molecular interactions of FAK activation and downstream events and analyze their functional role in integrin signaling using a cell migration model of transfected CHO cells. In Aim 1, the potential role of the Ras/MAP kinase pathway in FAK-stimulated cell migration will be investigated. Site-directed mutagenesis of FAK, along with dominant-negative src constructs, will be used to investigate the role of FAK association with src in regulating cell migration. Dominant-negative MEK constructs and inhibitors will also be used to correlate MAP kinase activation and cell migration in CHO cells expressing various FAK mutants. Similar structure/function studies will be performed to explore the role of the FAK-Grb2 association in regulating cell migration. Aim 2 will determine the role of other FAK downstream targets and substrates in cell migration. Potential downstream substrates of FAK in CHO cells overexpressing wild-type FAK will be identified, sites of phosphorylation of these substrates mapped, and their role in FAK-stimulated cell migration explored by mutagenesis and transfection assays. The final aim will determine the mechanism of FAK activation by integrins. The sequences on FAK that bind talin will be identified by site-directed mutagenesis and in vitro binding assays, and the potential functional significance of this interaction in mediating FAK activation by integrins and stimulation of cell migration will be investigated in CHO cell migration system. Other cellular proteins that bind to the b1integrin cytoplasmic domain will also be isolated by yeast two-hybrid screening, and their potential role in FAK activation will be explored by studying their subcellular localization, regulation of FAK activity and potential physical interactions with FAK. These studies will enhance our understanding of the molecular mechanisms of signal transduction by integrins, which are critical factors in cell migration and invasiveness.